| 1. |
- Rödström, Karin, et al.
(författare)
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Structure of the Superantigen Staphylococcal Enterotoxin B in Complex with TCR and Peptide-MHC Demonstrates Absence of TCR-Peptide Contacts.
- 2014
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 193:4, s. 1998-2004
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Tidskriftsartikel (refereegranskat)abstract
- Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells.
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| 2. |
- Elbing, Karin, 1974, et al.
(författare)
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Towards the structure-function-specificity relationship of glucose transporters
- 2010
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Ingår i: FEBS Journal, Special issue: Abstracts of the 35th FEBS Congress, Gothenburg, Sweden, 26 June - 1 July 2010. - 1742-464X. ; 277, supplement 1, s. 209-210
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Glucose plays a central role in nutrient sensing and signalling and its uptake through facilitated diffusion is mediated by membrane associated glucose transporters, GLUT proteins in mammals and Hxt proteins in the yeast S. cerevisiae. These proteins belong to the Major Facilitator Superfamily (MFS), which is present in all studied organisms. They transport a wide range of solutes such as amino acids, sugars, nucleotides, drugs, peptides, organic and inorganic anions, metabolites, neurotransmitters, polyols etc. All MFS proteins structurally investigated have been purified from their natural sources and successful heterologous production has not yet been reported. While there is a wealth of reports in the literature on mutagenesis studies, to analyze the structure-function relationship of glucose transporters, there are no three dimensional structures available. Our research goal is to achieve a more detailed understanding of the structure-function-specificity relationship of glucose transporters. To achieve this overall goal we have successfully expressed mammalian GLUTs and yeast Hxts in yeast. Initial solubilsation trials suggest that Brij35 solubilises GLUT4. Purification and crystallization analyses of the target proteins are ongoing.
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| 3. |
- Eriksson, Stefanie, et al.
(författare)
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NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:5
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Tidskriftsartikel (refereegranskat)abstract
- Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.
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| 4. |
- Palmgren, Madelene, et al.
(författare)
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Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins
- 2017
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Ingår i: Journal of Membrane Biology. - : Springer Science and Business Media LLC. - 0022-2631 .- 1432-1424. ; 250:6, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.
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| 5. |
- Söndergaard Hansen, Jesper, et al.
(författare)
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Glucose transport machinery reconstituted in cell models.
- 2015
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1364-548X .- 1359-7345. ; 51:12, s. 2316-2319
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Tidskriftsartikel (refereegranskat)abstract
- Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it.
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| 6. |
- Venskutonytė, Raminta, et al.
(författare)
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Expression and purification of rat glucose transporter 1 in Pichia pastoris
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1713
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Bokkapitel (refereegranskat)abstract
- Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.
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| 7. |
- Dinér, Peter, 1976, et al.
(författare)
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Short cut to 1,2,3-triazole-based p38 MAP kinase inhibitors via [3+2]-cycloaddition chemistry
- 2009
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Ingår i: New J. Chem.. - : Royal Society of Chemistry (RSC). - 1144-0546 .- 1369-9261. ; 33:5, s. 1010-1016
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Tidskriftsartikel (refereegranskat)abstract
- A series of 4,5-substituted 1,2,3-triazoles was synthesised via Cu(I)-catalysed azide–alkyne 1,3-dipolar [2+3]-cycloaddition reactions followed by a Suzuki coupling. The 1,2,3-triazoles were evaluated as inhibitors of the p38 MAP kinase, showing IC50 values in the high nanomolar range
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| 8. |
- Elbing, Karin, 1974
(författare)
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Differential Roles of the Beta Subunit Glycogen-Binding Domains in the Regulation of the Snf1 Kinase Complex.
- 2009
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Ingår i: Eukaryotic cell. - 1535-9786.
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Tidskriftsartikel (refereegranskat)abstract
- Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeast express three isoforms of the AMP-activated protein kinase consisting of the Snf1(alpha), Snf4 (gamma) and one of three alternative beta subunits, either Sip1, Sip2 or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen binding domain. Deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase as judged by a constitutive activity independent of glucose availability. In contrast, deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore the different Snf1 kinase isoforms are regulated through distinct mechanisms which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as Snf1 and casein kinase II. The significance of the phosphorylation is unclear since deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.
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| 9. |
- Elbing, Karin, 1974, et al.
(författare)
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Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae
- 2004
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:9, s. 5323-5330
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycollytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycollysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.
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| 10. |
- Elbing, Karin, 1974
(författare)
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The Role of Hexose Transport on Glycolytic Flux and Glucose Induced Responses in Saccharomyces cerevisiae
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the yeast Saccharomyces cerevisiae, glucose is primarily fermented rather than respired. Only in cultivation modes such as chemostats or fed-batches, where the external glucose is kept low, a fully respiratory catabolism is observed. We have constructed a series of yeast strains starting from a mutant (the hxt Null strain) that is unable to take up glucose. In these strains glucose uptake is governed by chimeras between the two hexose transporters Hxt1 and Hxt7. These strains display a broad range of glucose uptake capacities. They have been used to show that glucose uptake has a high degree of control on the glycolytic flux only when the uptake rate is significantly reduced. One of the strains, with a low uptake capacity, presented a completely Crabtree negative phenotype. This shows that a switch between fermentative and respiratory metabolism can be achieved by changing only a single metabolic step. The transferability of the phenotype has been confirmed by integration of the chimeric construct into a hxt1-hxt7 haploid wine strain. Strains with different glycolytic rates have also allowed quantitative studies on glucose-induced responses and dissection of different signaling mechanisms. A strong correlation between glycolytic rate and expression of glycolytic genes was observed. mRNA profiles of several groups of glucose-regulated genes suggest that sensing is primarily intracellular. Contrary to what is presently believed, SUC2 expression did not always correlate to high phosphorylation levels of Snf1 and Mig1, key regulators of glucose-controlled gene expression. These results suggest regulation either through additional pathway/s or independent regulation of the Snf1/Mig1 targeted kinases and phosphatases.
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